Metal ion binding properties and conformational states of calcium- and integrin-binding protein

被引:70
作者
Yamniuk, AP [1 ]
Nguyen, LT [1 ]
Hoang, TT [1 ]
Vogel, HJ [1 ]
机构
[1] Univ Calgary, Dept Biol Sci, Struct Biol Res Grp, Calgary, AB T2N 1N4, Canada
关键词
D O I
10.1021/bi035432b
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta3. The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca2+ ions in a sequential manner with dissociation constants (K-d) near 0.54 and 1.9 muM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg2+ ion to EF-3 with a K-d near 120 muM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.
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页码:2558 / 2568
页数:11
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